Antiangiogenic agents

ABSTRACT

Compositions and methods for treating mammalian disease characterized by undesirable angiogenesis by administering derivatives of 2-methoxyestradiol of the general formula:  
                 
wherein the variables are defined in the specification.

FIELD OF THE INVENTION

The present invention relates to treating disease states characterizedby abnormal cell mitosis and or abnormal angiogenesis. Moreparticularly, the present invention relates to certain analogs of2-methoxyestradiol (2ME2) and their effect on diseases characterized byabnormal cell mitosis and/or abnormal angiogenesis.

BACKGROUND OF THE INVENTION

As used herein, the term “angiogenesis” means the generation of newblood vessels into a tissue or organ. Under normal physiologicalconditions, humans or animals only undergo angiogenesis in very specificrestricted situations. For example, angiogenesis is normally observed inwound healing, fetal and embryonal development and formation of thecorpus luteum, endometrium and placenta. The control of angiogenesis isa highly regulated system of angiogenic stimulators and inhibitors. Thecontrol of angiogenesis has been found to be altered in certain diseasestates and, in many cases, the pathological damage associated with thedisease is related to the uncontrolled angiogenesis.

Both controlled and uncontrolled angiogenesis are thought to proceed ina similar manner. Endothelial cells and pericytes, surrounded by abasement membrane, form capillary blood vessels. Angiogenesis beginswith the erosion of the basement membrane by enzymes released byendothelial cells and leukocytes. The endothelial cells, which line thelumen of blood vessels, then protrude through the basement membrane.Angiogenic stimulants induce the endothelial cells to migrate throughthe eroded basement membrane. The migrating cells form a “sprout” offthe parent blood vessel, where the endothelial cells undergo mitosis andproliferate. The endothelial sprouts merge with each other to formcapillary loops, creating the new blood vessel. In the disease state,prevention of angiogenesis could avert the damage caused by the invasionof the new microvascular system.

Persistent, unregulated angiogenesis occurs in a multiplicity of diseasestates, tumor metastasis and abnormal growth by endothelial cells andsupports the pathological damage seen in these conditions. The diversepathological states created due to unregulated angiogenesis have beengrouped together as angiogenic dependent or angiogenic associateddiseases. Therapies directed at control of the angiogenic processescould lead to the abrogation or mitigation of these diseases.

One example of a disease mediated by angiogenesis is ocular neovasculardisease. This disease is characterized by invasion of new blood vesselsinto the structures of the eye such as the retina or cornea. It is themost common cause of blindness and is involved in approximately twentyeye diseases. In age-related macular degeneration, the associated visualproblems are caused by an ingrowth of chorioidal capillaries throughdefects in Bruch's membrane with proliferation of fibrovascular tissuebeneath the retinal pigment epithelium. Angiogenic damage is alsoassociated with diabetic retinopathy, retinopathy of prematurity,corneal graft rejection, neovascular glaucoma and retrolentalfibroplasia. Other diseases associated with corneal neovascularizationinclude, but are not limited to, epidemic keratoconjunctivitis, VitaminA deficiency, contact lens overwear, atopic keratitis, superior limbickeratitis, pterygium keratitis sicca, sjogrens, acne rosacea,phylectenulosis, syphilis, Mycobacteria infections, lipid degeneration,chemical burns, bacterial ulcers, fungal ulcers, Herpes simplexinfections, Herpes zoster infections, protozoan infections, Kaposisarcoma, Mooren ulcer, Terrien's marginal degeneration, mariginalkeratolysis, rheumatoid arthritis, systemic lupus, polyarteritis,trauma, Wegeners sarcoidosis, Scleritis, Steven's Johnson disease,periphigoid radial keratotomy, and corneal graph rejection.

Diseases associated with retinal/choroidal neovascularization include,but are not limited to, diabetic retinopathy, macular degeneration,sickle cell anemia, sarcoid, syphilis, pseudoxanthoma elasticum, Pagetsdisease, vein occlusion, artery occlusion, carotid obstructive disease,chronic uveitis/vitritis, mycobacterial infections, Lyme's disease,systemic lupus erythematosis, retinopathy of prematurity, Eales disease,Bechets disease, infections causing a retinitis or choroiditis, presumedocular histoplasmosis, Bests disease, myopia, optic pits, Stargartsdisease, pars planitis, chronic retinal detachment, hyperviscositysyndromes, toxoplasmosis, trauma and post-laser complications. Otherdiseases include, but are not limited to, diseases associated withrubeosis (neovasculariation of the angle) and diseases caused by theabnormal proliferation of fibrovascular or fibrous tissue including allforms of proliferative vitreoretinopathy.

Another disease in which angiogenesis is believed to be involved isrheumatoid arthritis. The blood vessels in the synovial lining of thejoints undergo angiogenesis. In addition to forming new vascularnetworks, the endothelial cells release factors and reactive oxygenspecies that lead to pannus growth and cartilage destruction. Thefactors involved in angiogenesis may actively contribute to, and helpmaintain, the chronically inflamed state of rheumatoid arthritis.

Factors associated with angiogenesis may also have a role inosteoarthritis. The activation of the chondrocytes by angiogenic-relatedfactors contributes to the destruction of the joint. At a later stage,the angiogenic factors would promote new bone formation. Therapeuticintervention that prevents the bone destruction could halt the progressof the disease and provide relief for persons suffering with arthritis.

Chronic inflammation may also involve pathological angiogenesis. Suchdisease states as ulcerative colitis and Crohn's disease showhistological changes with the ingrowth of new blood vessels into theinflamed tissues. Bartonellosis, a bacterial infection found in SouthAmerica, can result in a chronic stage that is characterized byproliferation of vascular endothelial cells. Another pathological roleassociated with angiogenesis is found in atherosclerosis. The plaquesformed within the lumen of blood vessels have been shown to haveangiogenic stimulatory activity.

One of the most frequent angiogenic diseases of childhood is thehemangioma. In most cases, the tumors are benign and regress withoutintervention. In more severe cases, the tumors progress to largecavernous and infiltrative forms and create clinical complications.Systemic forms of hemangiomas, the hemangiomatoses, have a highmortality rate. Therapy-resistant hemangiomas exist that cannot betreated with therapeutics currently in use.

Angiogenesis is also responsible for damage found in hereditary diseasessuch as Osler-Weber-Rendu disease, or hereditary hemorrhagictelangiectasia. This is an inherited disease characterized by multiplesmall angiomas, tumors of blood or lymph vessels. The angiomas are foundin the skin and mucous membranes, often accompanied by epistaxis(nosebleeds) or gastrointestinal bleeding and sometimes with pulmonaryor hepatic arteriovenous fistula.

Angiogenesis is prominent in solid tumor formation and metastasis.Angiogenic factors have been found associated with several solid tumorssuch as rhabdomyosarcomas, retinoblastoma, Ewing sarcoma, neuroblastoma,and osteosarcoma. A tumor cannot expand without a blood supply toprovide nutrients and remove cellular wastes. Tumors in whichangiogenesis is important include solid tumors, and benign tumors suchas acoustic neuroma, neurofibroma, trachoma and pyogenic granulomas.Prevention of angiogenesis could halt the growth of these tumors and theresultant damage to the animal due to the presence of the tumor.

It should be noted that angiogenesis has been associated with blood-borntumors such as leukemias, any of various acute or chronic neoplasticdiseases of the bone marrow in which unrestrained proliferation of whiteblood cells occurs, usually accompanied by anemia, impaired bloodclotting, and enlargement of the lymph nodes, liver, and spleen. It isbelieved that angiogenesis plays a role in the abnormalities in the bonemarrow that give rise to leukemia-like tumors.

Angiogenesis is important in two stages of tumor metastasis. The firststage where angiogenesis stimulation is important is in thevascularization of the tumor which allows tumor cells to enter the bloodstream and to circulate throughout the body. After the tumor cells haveleft the primary site, and have settled into the secondary, metastasissite, angiogenesis must occur before the new tumor can grow and expand.Therefore, prevention of angiogenesis could lead to the prevention ofmetastasis of tumors and possibly contain the neoplastic growth at theprimary site.

Knowledge of the role of angiogenesis in the maintenance and metastasisof tumors has led to a prognostic indicator for breast cancer. Theamount of neovascularization found in the primary tumor was determinedby counting the microvessel density in the area of the most intenseneovascularization in invasive breast carcinoma. A high level ofmicrovessel density was found to correlate with tumor recurrence.Control of angiogenesis by therapeutic means could possibly lead tocessation of the recurrence of the tumors.

Angiogenesis is also involved in normal physiological processes such asreproduction and wound healing. Angiogenesis is an important step inovulation and also in implantation of the blastula after fertilization.Prevention of angiogenesis could be used to induce amenorrhea, to blockovulation or to prevent implantation by the blastula.

In wound healing, excessive repair or fibroplasia can be a detrimentalside effect of surgical procedures and may be caused or exacerbated byangiogenesis. Adhesions are a frequent complication of surgery and leadto problems such as small bowel obstruction.

Several kinds of compounds have been used to prevent angiogenesis.Taylor et al. have used protamine to inhibit angiogenesis, see Taylor etal., Nature 297:307 (1982). The toxicity of protamine limits itspractical use as a therapeutic. Folkman et al. have disclosed the use ofheparin and steroids to control angiogenesis. See Folkman et al.,Science 221:719 (1983) and U.S. Pat. Nos. 5,001,116 and 4,994,443.Steroids, such as tetrahydrocortisol, which lack gluco and mineralcorticoid activity, have been found to be angiogenic inhibitors.

Other factors found endogenously in animals, such as a 4 kDaglycoprotein from bovine vitreous humor and a cartilage derived factor,have been used to inhibit angiogenesis. Cellular factors such asinterferon inhibit angiogenesis. For example, interferon α or humaninterferon β has been shown to inhibit tumor-induced angiogenesis inmouse dermis stimulated by human neoplastic cells. Interferon β is alsoa potent inhibitor of angiogenesis induced by allogeneic spleen cells.See Sidky et al., Cancer Research 47:5155-5161 (1987). Human recombinantα interferon (alpha/A) was reported to be successfully used in thetreatment of pulmonary hemangiomatosis, an angiogenesis-induced disease.See White et al., New England J. Med. 320:1197-1200 (1989).

Other agents which have been used to inhibit angiogenesis includeascorbic acid ethers and related compounds. See Japanese Kokai TokkyoKoho No. 58-131978. Sulfated polysaccharide DS 4152 also showsangiogenic inhibition. See Japanese Kokai Tokkyo Koho No. 63-119500. Afungal product, fumagillin, is a potent angiostatic agent in vitro. Thecompound is toxic in vivo, but a synthetic derivative, AGM 12470, hasbeen used in vivo to treat collagen II arthritis. Fumagmin andO-substituted fumagillin derivatives are disclosed in EPO PublicationNos. 0325199A2 and 0357061A1. Folkman et al., described several proteinsderived from endogenous proteins including angiostatin and endostatin.(See, for example, U.S. Pat. Nos. 6,024,688 and 5,854,205 which areincorporated in their entirety) D'Amato et al., described2-methoxyestradiol and derivatives of 2-methoxyestradiol in U.S. Pat.Nos. 5,504,074 and 5,661,143 which are incorporated herein by referenceentirety.

The above compounds are either topical or injectable therapeutics.Therefore, there are drawbacks to their use as a general angiogenicinhibitor and lack adequate potency. For example, in prevention ofexcessive wound healing, surgery on internal body organs involvesincisions in various structures contained within the body cavities.These wounds are not accessible to local applications of angiogenicinhibitors. Local delivery systems also involve frequent dressings whichare impracticable for internal wounds, and increase the risk ofinfection or damage to delicate granulation tissue for surface wounds.

Thus, a method and composition are needed that are capable of inhibitingangiogenesis and which are easily administered. A simple and efficaciousmethod of treatment would be through the oral route. If an angiogenicinhibitor could be given by an oral route, the many kinds of diseasesdiscussed above, and other angiogenic dependent pathologies, could betreated easily. The optimal dosage could be distributed in a form thatthe patient could self-administer.

Other diseases are also characterized by an abnormal balance betweencellular mitosis and apoptosis. One of these diseases is osteoporosis.Osteoporosis is characterized by a reduction in the bone mass of theskeleton which leads to skeletal fragility and an increased risk offracture. In humans, the most common sites of fracture are found in theforearm, the vertebrae and the hip bones. Osteoporosis and its attendantfractures are a major cause of morbidity and mortality and lead toincreased health costs for care.

In treating osteoporosis the main objective is to prevent fractures bystopping the loss of skeletal integrity. A variety of differenttherapies have been tried to achieve this objective, such as calcium,Vitamin D supplements and hormone replacement. Calcitonin has been usedto improve bone mineral density at all bone sites. Bisphosphonates arean important group of therapeutic agents used for treatment ofosteoporosis. They act by inhibiting bone resorption and increase bonedensity. Cyclical etidronate treatment aids in decreasing vertebralfractures, as does hormone replacement therapy and calcitonin.Alendronate has been shown to decrease the risk of symptomatic fracturesof the forearm, spine and hip.

None of these treatments have proven to be effective in large numbers ofosteoporotic patients. Additionally, the currently used therapies haveunwanted side effects that create compliance and tolerance problems intreatment regimens. The most common adverse events with cyclicaletidronate and alendronate are gastrointestinal disturbances.Esophagitis has also been a complication of therapies with alendronate.Cyclical etidronate has been shown to lead to focal osteomalacia.Hormone replacement therapies lead to estrogen effects such as uterinehypertrophy, and a potential for stimulation of estrogen-sensitivetumors leading to complications such as breast cancer.

What is needed are safe and effective treatments that do not createunwanted side effects.

2-Methoxyestradiol is an endogenous metabolite of estradiol (E2) thathas potent anti-proliferative activity and induces apoptosis in a widevariety of tumor and non-tumor cell lines. When administered orally, itexhibits anti-tumor and anti-proliferative activity with little or notoxicity. In vitro data suggests that 2-methoxyestradiol does not engagethe estrogen receptor for its anti-proliferative activity and is notestrogenic over a wide range of concentrations, as accessed by estrogendependant MCF-7 cell proliferation. However, the presence ofdemethylases in vivo may metabolize this compound to 2-hydroxyestradiol,which has been shown to be estrogenic by several approaches. What isneeded is a means to improve the bioavailibility of estradiol or2-methoxyestradiol and to reduce the formation of estrogenic2-methoxyestradiol metabolities. What is also needed is a means tomodify estradiol or 2-methoxyestradiol in such a way that the moleculecan not be converted into an uterotropic derivative.

SUMMARY OF THE INVENTION

The present invention provides certain analogs of 2-methoxyestradiolthat are effective in treating diseases characterized by abnormalmitosis and/or abnormal angiogenesis. Specifically the present inventionrelates to analogs of 2-methoxyestradiol that have been modified at the2 position and the 16 position. Compounds within the general formulaethat inhibit cell proliferation are preferred. Preferred compositionsmay also exhibit a change (increase or decrease) in estrogen receptorbinding, improved absorption, transport (e.g. through blood-brainbarrier and cellular membranes), biological stability, or decreasedtoxicity. The invention also provides compounds useful in the method, asdescribed by the general formulae of the claims.

A mammalian disease characterized by undesirable cell mitosis, asdefined herein, includes but is not limited to excessive or abnormalstimulation of endothelial cells (e.g., atherosclerosis), solid tumorsand tumor metastasis, benign tumors, for example, hemangiomas, acousticneuromas, neurofibromas, trachomas, and pyogenic granulomas, vascularmalfunctions, abnormal wound healing, inflammatory and immune disorders,Bechet's disease, gout or gouty arthritis, abnormal angiogenesisaccompanying: rheumatoid arthritis, psoriasis, diabetic retinopathy, andother ocular angiogenic diseases such as retinopathy of prematurity(retrolental fibroplasic), macular degeneration, comeal graft rejection,neovascular glaucoma and Osler Weber syndrome. Other undesiredangiogenesis involves normal processes including ovulation andimplantation of a blastula Accordingly, the compositions described abovecan be used to block ovulation and implantation of a blastula or toblock menstruation (induce amenorrhea).

Since 2-methoxyestradiol is metabolized to a much less activemetabolite, the present invention adds steric bulk and/or modificationof electrostatic characteristics at position 16 of 2-methoxyestradiolfor retarding or preventing interaction of 17β-hydroxysteroiddehydrogenases and co-factor NADP⁺ on this substrate. Addition of stericbulk and/or modification of electrostatic characteristics at position 16of 2-methoxyestradiol may retard or prevent glucuronidation. It isbelieved that retardation or prevention of these two metabolicdeactivation pathways prolongs the serum lifetime of 2-methoxyestradioland other estrogenic compounds while retaining the desiredanti-angiogenic and anti-tumor activity.

Aside from preventing the possible metabolism of 2ME2 to 2ME1, which mayoccur by making these steroids poor substrates for 17B-HSD (by eithersteric and/or electronic effects), it is not possible for these analogsto undergo the demethylation known to occur with 2ME2 since there is nomethyl ether group at that position. This is desirable since it has beendemonstrated that 2-hydroxyestradiol (the product of demethylation of2ME2) has estrogenic activity.

Also disclosed is a method for modifying the methyl ether of2-methoxyestradiol so that it can not be a substrate for demethylase andthe resulting compounds.

Other features and advantages of the invention will be apparent from thefollowing description of preferred embodiments thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts: I. colchicine, 2-methoxyestradiol and combretastatinA-4, and II. various estradiol derivatives comprising colchicine (a-c)or combretastatin A-4 (d) structural motifs as described below.

DETAILED DESCRIPTION OF THE INVENTION

As described below, compounds that are useful in accordance with theinvention include novel estradiol derivatives that exhibit anti-mitotic,anti-angiogenic and anti-tumor properties. Specific compounds accordingto the invention are described below. Preferred compounds of theinvention are estradiol derivatives modified at either the 2 or 16positions. Those skilled in the art will appreciate that the inventionextends to other compounds within the formulae given in the claimsbelow, having the described characteristics. These characteristics canbe determined for each test compound using the assays detailed below andelsewhere in the literature.

Without wishing to be bound to specific mechanisms or theory, it appearsthat certain compounds that are known to exhibit anti-mitotic propertiessuch as colchicine and combretastatin A-4 share certain structuralsimilarities with estradiol. FIG. 1 illustrates the molecular formulaeof estradiol, colchicine, combretastatin A-4, and improved estradiolderivatives that exhibit anti-mitotic, anti-angiogenic and anti-tumorproperties. Molecular formulae are drawn and oriented to emphasizestructural similarities between the ring structures of colchicine,combretastatin A-4, estradiol, and certain estradiol derivatives.Estradiol derivatives are made by incorporating colchicine orcombretastatin A-4 structural motifs into the steroidal backbone ofestradiol.

FIG. 1, part I, depicts the chemical formulae of colchicine,2-methoxyestradiol and combretastatin A-4. FIG. 1, part II a-d,illustrates estradiol derivatives that comprise structural motifs foundin colchicine or combretastatin A-4. For example, part II a-c showsestradiol derivatives with an A and/or B ring expanded from six to sevencarbons as found in colchicine and part Ild depicts an estradiolderivative with a partial B ring as found in combretastatin A-4. Each Cring of an estradiol derivative, including those shown in FIG. 1, may befully saturated as found in 2-methoxyestradiol. R₁₋₆ represent a subsetof the substitution groups found in the claims. Each R₁₋R₆ canindependently be defined as —R₁, OR₁, —OCOR₁₁—SR₁, —F, —NHR₂, —Br, —I,or —C≡CH.

2-Methoxyestradiol is an endogenous metabolite of estradiol that haspotent anti-proliferative activity and induces apoptosis in a widevariety of tumor and non-tumor cell lines. When administered orally, itexhibits anti-tumor and anti-proliferative activity with little or notoxicity. 2-Methoxyestradiol is metabolized to a much less activemetabolite, 2-methoxyestrone as indicated by in vitro and in vivoresults. Although not wishing to be bound by theory, it is believed thatthis metabolite is formed through the same enzymatic pathway as estroneis formed from estradiol. Although not wishing to be bound by theory, itis believed that the enzymes responsible for this reversible reaction onestradiol are the 17β-hydroxysteroid dehydrogenases (17β-HSD) and NADP+co-factor (Han et al., J. Biol. Chem. 275:2, 1105-1111 (Jan. 12, 2000)and other references cited earlier). Each of the four members of thisenzyme family, types 1, 2, 3, and 4, have distinct activity. It appearsthat 17β-HSD type 1 catalyzes the reductive reaction (estrone toestradiol), while 17β-HSD type 2 catalyzes the oxidation reaction(estradiol to estrone), and type 3 catalyzes 4-androstenedione totestosterone. An additional metabolic deactivation pathway results inglucuronidation of 2-methoxyestradiol.

Since 2-methoxyestradiol is metabolized to a much less activemetabolite, the present invention adds steric bulk and/or modificationof electrostatic characteristics at position 16 of 2-methoxyestradiolfor retarding or preventing interaction of the family of17β-hydroxysteroid dehydrogenases and co-factor NADP⁺ on this substrate.Addition of steric bulk and/or modification of electrostaticcharacteristics at position 16 of 2-methoxyestradiol also retards orprevents glucuronidation. It is believed that retardation or preventionof these two metabolic deactivation pathways prolongs the serum lifetimeof 2-methoxyestradiol and other estradiol derivatives while retainingthe desired anti-angiogenic and anti-tumor activity.

Aside from preventing the possible metabolism of 2ME2 to 2ME1, which mayoccur by making these steroids poor substrates for 17B-HSD (by eithersteric and/or electronic effects), it is not possible for these analogsto undergo the demethylation known to occur with 2ME2 since there is nomethyl ether group at that position. This is desirable since it has beendemonstrated that 2-hydroxyestradiol (the product of demethylation of2ME2) has estrogenic activity.

In another embodiment of the invention, estradiol derivatives aremodified at the 2 position.

Anti-Proliferative Activity In Situ

Anti-proliferative activity is evaluated in situ by testing the abilityof an improved estradiol derivative to inhibit the proliferation of newblood vessel cells (angiogenesis). A suitable assay is the chick embryochorioallantoic membrane (CAM) assay described by Crum et al. Science230:1375 (1985). See also, U.S. Pat. No. 5,001,116, hereby incorporatedby reference, which describes the CAM assay. Briefly, fertilized chickembryos are removed from their shell on day 3 or 4, and amethylcellulose disc containing the drug is implanted on thechorioallantoic membrane. The embryos are examined 48 hours later and,if a clear avascular zone appears around the methylcellulose disc, thediameter of that zone. is measured. Using this assay, a 100 mg disk ofthe estradiol derivative 2-methoxyestradiol was found to inhibit cellmitosis and the growth of new blood vessels after 48 hours. This resultindicates that the anti-mitotic action of 2-methoxyestradiol can inhibitcell mitosis and angiogenesis.

Anti-Proliferative Activity In Vitro

The process by which 2ME₂ affects cell growth remains unclear, however,a number of studies have implicated various mechanisms of action andcellular targets. 2ME₂ induced changes in the levels and activities ofvarious proteins involved in the progression of the cell cycle. Theseinclude cofactors of DNA replication and repair, e.g., proliferatingcell nuclear antigen (PCNA) (Klauber, N., Parangi, S., Flynn, E., Hamel,E. and D'Amato, R. J. (1997), Inhibition of angiogenesis and breastcancer in mice by the nicrotubule inhibitors 2-methoxyestradiol andTaxol., Cancer Research 57, 81-86; Lottering, M-L., de Kock, M.,Viljoen, T. C., Grobler, C. J. S. and Seegers, J. C. (1996)17β-estradiol metabolites affect some regulators of the MCF-7 cellcycle. Cancer Letters 110, 181-186); cell division cycle kinases andregulators, e.g., p34^(cdc2) and cyclin B (Lottering et al. (1996);Attalla, H., Mäkelä, T. P., Adlercreutz, H. and Andersson, L. C. (1996)2-methoxyestradiol arrests cells in mitosis without depolymerizingtubulin. Biochemnical and Biophysical Research Communications 228,467-473; Zoubine, M. N., Weston, A. P., Johnson, D. C., Campbell, D. R.and Banerjee, S. K. (1999) 2-Methoxyestradiol-induced growth suppressionand lethality in estrogen-responsive MCF-7 cells may be mediated by downregulation of p34cdc2 and cyclin B1 expression. Int J Oncol 15,639-646); transcription factor modulators, e.g., SAPK/JNK (Yue, T-L.,Wang, X., Louden, C. S., Gupta, L. S., Pillarisetti, K., Gu, J-L., Hart,T. K., Lysko, P. G. and Feuerstein, G. Z. (1997) 2-methoxyestradiol, anendogenous estrogen metabolite induces apoptosis in endothelial cellsand inhibits angiogenesis: Possible role for stress-activated proteinkinase signaling pathway and fas expression. Molecular Pharmacology 51,951-962; Attalla, H., Westberg, J. A., Andersson, L. C., Aldercreutz, H.and Makela, T. P. (1998) 2-Methoxyestradiol-induced phosphorylation ofbcl-2: uncoupling from JNK/SAPK activation. Biochem and Biophys ResCommun 247, 616-619); and regulators of cell arrest and apoptosis, e.g.,tubulin (D'Amato, R. J., Lin, C. M., Flynn, E., Folkman, J. and Hamel,E. (1994) 2-Methoxyestradiol, and endogenous mammalian metabolite,inhibits tubulin polymerization by interacting at the colchicine site.Proc. Natl. Acad. Sci. USA 91, 3964-3968; Hamel, E., Lin, C. M., Flynn,E. and D'Amato, R. J. (1996) Interactions of 2-methoxyestradiol, andendogenous mammalian metabolite, with unploymerized tubulin and withtubulin polymers. Biochemistry 35, 1304-1310), P21^(WAF1/C1P1)(Mukhopadhyay, T. and Roth, J. A. (1997) Induction of apoptosis in humanlung cancer cells after wild-type p53 activation by methoxyestradiol.Oncogene 14, 379-384), bcl-2 and FAS (Yue et al. (1997); Attalla et al.(1998)), and p53 (Kataoka, M., Schumacher, G., Cristiano, R. J.,Atkinson, E. N., Roth, J. A. and Mukhopadhyay, T. (1998) An agent thatincreases tumor suppressor transgene product coupled with systemictransgene delivery inhibits growth of metastatic lung cancer in vivo.Cancer Res 58, 47614765; Mukhopadhyay et al. (1997); Seegers, J. C.,Lottering, M-L., Grobler C. J. S., van Papendorp, D. H., Habbersett, R.C., Shou, Y. and Lehnert B. E. (1997) The mammalian metabolite,2-methoxyestradiol, affects p53 levels and apoptosis induction intransformed cells but not in normal cells. J. Steroid Biochem. Molec.Biol. 62, 253-267). The effects on the level of cAMP, calmodulinactivity and protein phosphorylation may also be related to each other.More recently, 2ME2 was shown to upregulate Death Receptor 5 and caspase8 in human endothelial and tumor cell lines (LaVallee, T. M., Hembrough,W. A., Williams, M. S., Zhan, X. H., Pribluda, V. S., Papathanassiu, A.,and Green, S. J. 2-Methoxyestradiol upregulates DR5 and inducesapoptosis independently of p53. (Submitted for publication)). Allcellular targets described above are not necessarily mutually exclusiveto the inhibitory effects of 2ME2 in actively dividing cells.

The high affinity binding to SHBG. has been mechanistically associatedto its efficacy in a canine model of prostate cancer, in which signalingby estradiol and 5α-androstan-3α,17β-diol were inhibited by 2ME₂ (Ding,V. D., Moller, D. E., Feeney, W. P., Didolkar, V., Nakhla, A. M.,Rhodes, L., Rosner, W. and Smith, R. G. (1998) Sex hormone-bindingglobulin mediates prostate androgen receptor action via a novelsignaling pathway. Endocrinology 139, 213-218).

The more relevant mechanism described above have been extensivelydiscussed in Victor S. Pribluda, Theresa M. LaVallee and Shawn J. Green,2-methoxyestradiol: a novel endogenous chemotherapeutic andantiangiogenic in The New Angiotherapy, Tai-Ping Fan and Robert Auerbacheds., Human Press Publisher.

Assays relevant to the mechanisms of action and activity are well-knownin the art. For example, anti-mitotic activity mediated by effects ontubulin polymerization activity can be evaluated by testing the abilityof an estradiol derivative to inhibit tubulin polymerization andmicrotubule assembly in vitro. Microtubule assembly is followed in aGilford recording spectrophotometer (model 250 or 2400S) equipped withelectronic temperature controllers. A reaction mixture (allconcentrations refer to a final reaction volume of 0.25 μl ) contains1.0M monosodium glutamate (pH 6.6), 1. omg/ml (10 μM) tubulin, 1.0 mMMgCl₂, 4% (v/v) dimethlylsulfoxide and 20-75 μM of a composition to betested. The 0.24 ml reaction mixtures are incubated for 15 min. at 37°C. and then chilled on ice. After addition of 10 μl 2.5 mM GTP, thereaction mixture is transferred to a cuvette at 0° C., and a baselineestablished. At time zero, the temperature controller of thespectrophotometer is set at 37° C. Microtubule assembly is evaluated byincreased turbity at 350 nm. Alternatively, inhibition of microtubuleassembly can be followed by transmission electron microscopy asdescribed in Example 2 below.

Other such assays include counting of cells in tissue culture plates orassessment of cell number through metabolic assays or incorporation intoDNA of labeled (³H-thymidine) or immuno-reactive (BrdU) nucleotides. Inaddition, antiangiogenic activity may be evaluated through endothelialcell migration, endothelial cell tubule formation, or vessel outgrowthin ex-vivo models such as rat aortic rings.

Indications

The invention can be used to treat any disease characterized by abnormalcell mitosis. Such diseases include, but are not limited to: abnormalstimulation of endothelial cells (e.g., atherosclerosis), solid tumorsand tumor metastasis, benign tumors, for example, hemangiomas, acousticneuromas, neurofribomas, trachomas, and pyogenic granulomas, vascularmalfunctions, abnormal wound healing, inflammatory and immune disorders,Bechet's disease, gout or gouty arthritis, abnormal angiogenesisaccompanying: rheumatoid arthritis, psoriasis, diabetic retinopathy, andother ocular angiogenic diseases such as retinopathy of prematurity(retrolental fibroplasic), macular degeneration, corneal graftrejection, neuroscular glacoma and Oster Webber syndrome.

In addition, the invention can be used to treat a variety ofpost-menapausal symptoms, including osteoporosis, cardiovasculardisease, Alzheimer's disease, to reduce the incidence of strokes, and asan alternative to prior estrogen replacement therapies. The compounds ofthe present invention can work by estrogenic and non-estrogenicbiochemical pathways.

Improved Estradiol Derivative Synthesis

Known compounds that are used in accordance with the invention andprecursors to novel compounds according to the invention can bepurchased, e.g., from Sigma Chemical Co., St. Louis, Steraloids andResearch Plus. Other compounds according to the invention can besynthesized according to known methods from publicly availableprecursors.

The chemical synthesis of estradiol has been described (Eder, V. et al.,Ber 109, 2948 (1976); Oppolzer, D. A. and Roberts, D A. Helv. Chim.Acta. 63, 1703, (1980)). Synthetic methods for making seven-memberedrings in multi-cyclic compounds are known (Nakamuru, T. et al. Chem.Pharm. Bull. 10, 281 (1962); Sunagawa, G. et al. Chem. Pharm. Bull. 9,81 (1961); Van Tamelen, E. E. et al. Tetrahedren 14, 8-34 (1961); Evans,D. E. et al. JACS 103, 5813 (1981)). Those skilled in the art willappreciate that the chemical synthesis of estradiol can be modified toinclude 7-membered rings by making appropriate changes to the startingmaterials, so that ring closure yields seven-membered rings. Estradiolor estradiol derivatives can be modified to include appropriate chemicalside groups according to the invention by known chemical methods (TheMerck Index, 11th Ed., Merck & Co., Inc., Rahway, N.J. USA (1989), pp.583-584).

Analogs of 2ME2 or 2-ethoxyestradiol containing 7 membered rings can bemodified to include appropriate chemical side groups according to theinvention by known chemical methods (see for example, Miller, T. A.;Bulman, A. L.; Thompson, C. D.; Garst, M. E.; Macdonald, T. L.“Synthesis and Structure-Activity Profiles of A-Homoestranes, theEstratropones.” J. Med. Chem., 1997, 40, 3836-3841; Miller, T. A.;Bulman, A. L.; Thompson, C. D.; Garst, M. E.; Macdonald, T. L. “TheSynthesis and Evaluation of Functionalized Estratropones-PotentInhibitors of Tubulin Polymerization.” Bioorg. Med. Chem. Letters, 1997,7, 1851-1856; and Wang, Z.; Yang, D.; Mohanakrishnan, A. K.; Fanwick, P.E.; Nampoothiri, P.; Hamel, E.; Cushman, M. “Synthesis of B-RingHomologated Estradiol Analogs that Modulate Tubulin Polymerization andMicrotubule Stability.” J. Med. Chem., 2000, 43, 2419-2429. Thesearticles do not utilize ring closure strategies to make the sevenmembered ring, rather they use a ring expansion strategy. The Cushmanarticle explores B-Ring expanded analogs whereas the other articles dealwith the expanded the A-ring.)

The synthetic pathways used to prepare the derivatives of the presentinvention are based on modified published literature procedures forestradiol derivatives and dimethylenamines (Trembley et al., Bioorganic& Med. Chem. 1995 3, 505-523; Fevig et al:, J. Org. Chem., 1987 52,247-251; Gonzalez et al., Steroids 1982, 40, 171-187; Trembley et al.,Synthetic Communications 1995, 25, 2483-2495; Newkome et al., J. Org.Chem. 1966, 31, 677-681; Corey et al Tetrahedron Lett 1976, 3-6; andCorey et. al., Tetrahedron Lett, 1976, 3667-3668]. The modifications areprovided in Example 1 below. Initial screening of epimeric16-ethyl-2-methoxyestradiol and related analogues showed that it isabout equipotent to 2-methoxyestradiol in inhibition of HUVEC cellproliferation in vitro.

Administration

The compositions described above can be provided as physiologicallyacceptable formulations using known techniques, and these formulationscan be administered by standard routes. In general, the combinations maybe administered by the topical, oral, rectal or parenteral (e.g.,intravenous, subcutaneous or intramuscular) route. In addition, thecombinations may be incorporated into biodegradable polymers allowingfor sustained release, the polymers being implanted in the vicinity ofwhere delivery is desired, for example, at the site of a tumor. Thebiodegradable polymers and their use are described in detail in Brem etal., J. Neurosurg. 74:441-446 (1991). The dosage of the composition willdepend on the condition being treated, the particular derivative used,and other clinical factors such as weight and condition of the patientand the route of administration of the compound. However, for oraladministration to humans, a dosage of 0.01 to 100 mg/kg/day, preferably0.01-1 mg/kg/day, is generally sufficient.

The formulations include those suitable for oral, rectal, nasal, topical(including buccal and sublingual), vaginal or parenteral (includingsubcutaneous, intramuscular, intravenous, intradermal, intraocular,intratracheal, and epidural) administration. The formulations mayconveniently be presented in unit dosage form and may be prepared byconventional pharmaceutical techniques. Such techniques include the stepof bringing into association the active ingredient and thepharmaceutical carrier(s) or excipient(s). In general, the formulationsare prepared by uniformly and intimately bringing into associate theactive ingredient with liquid carriers or finely divided solid carriersor both, and then, if necessary, shaping the product.

Formulations of the present invention suitable for oral administrationmay be presented as discrete units such as capsules, cachets or tabletseach containing a predetermined amount of the active ingredient; as apowder or granules; as a solution or a suspension in an aqueous liquidor a non-aqueous liquid; or as an oil-in-water liquid emulsion or awater-in-oil emulsion and as a bolus, etc.

A tablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared bycompressing, in a suitable machine, the active ingredient in afree-flowing form such as a powder or granules, optionally mixed with abinder, lubricant, inert diluent, preservative, surface-active ordispersing agent. Molded tables may be made by molding, in a suitablemachine, a mixture of the powdered compound moistened with an inertliquid diluent. The tablets may optionally coated or scored and may beformulated so as to provide a slow or controlled release of the activeingredient therein.

Formulations suitable for topical administration in the mouth includelozenges comprising the ingredients in a flavored basis, usually sucroseand acacia or tragacanth; pastilles comprising the active ingredient inan inert basis such as gelatin and glycerin, or sucrose and acacia; andmouthwashes comprising the ingredient to be administered in a suitableliquid carrier.

Formulations suitable for topical administration to the skin may bepresented as ointments, creams, gels and pastes comprising theingredient to be administered in a pharmaceutical acceptable carrier. Apreferred topical delivery system is a transdermal patch containing theingredient to be administered.

Formulations for rectal administration may be presented as a suppositorywith a suitable base comprising, for example, cocoa butter or asalicylate.

Formulations suitable for nasal administration, wherein the carrier is asolid, include a coarse powder having a particle size, for example, inthe range of 20 to 500 microns which is administered in the manner inwhich snuff is taken, i.e., by rapid inhalation through the nasalpassage from a container of the powder held close up to the nose.Suitable formulations, wherein the carrier is a liquid, foradministration, as for example, a nasal spray or as nasal drops, includeaqueous or oily solutions of the active ingredient.

Formulations suitable for vaginal administration may be presented aspessaries, tampons, creams, gels, pastes, foams or spray formulationscontaining in addition to the active ingredient such as carriers as areknown in the art to be appropriate.

Formulations suitable for parenteral administration include aqueous andnon-aqueous sterile injection solutions which may contain anti-oxidants,buffers, bacteriostats and solutes which render the formulation isotonicwith the blood of the intended recipient; and aqueous and non-aqueoussterile suspensions which may include suspending agents and thickeningagents. The formulations may be presented in unit-dose or multi-dosecontainers, for example, sealed ampules and vials, and may be stored ina freeze-dried (lyophilized) conditions requiring only the addition ofthe sterile liquid carrier, for example, water for injections,immediately prior to use. Extemporaneous injection solutions andsuspensions may be prepared from sterile powders, granules and tables ofthe kind previously described.

Preferred unit dosage formulations are those containing a daily dose orunit, daily sub-dose, as herein above recited, or an appropriatefraction thereof, of the administered ingredient.

2-Methoxyestradiol is an endogenous metabolite of estradiol (E2) thathas potent anti-proliferative activity and induces apoptosis in a widevariety of tumor and non-tumor cell lines. When administered orally, itexhibits anti-tumor and anti-proliferative activity with little or notoxicity. In vitro data suggests that 2-methoxyestradiol does not engagethe estrogen receptor for its anti-proliferative activity and is notestrogenic over a wide range of concentrations, as accessed by estrogendependant MCF-7 cell proliferation. However, the presence ofdemethylases in vivo may metabolize this compound to 2-hydroxyestradiol,which has been shown to be estrogenic by several approaches. The presentinvention improves the bioavallibility of estradiol or2-methoxyestradiol and to reduces the formation of estrogenic2-methoxyestradiol metabolities. The present invention modifiesestradiol or 2-methoxyestradiol in such a way that the molecule can notbe converted into an uterotropic derivative.

One embodiment of the invention modifies the methyl ether of2-methoxyestradiol so that it can not be a substrate for demethylase.Additionally, it has been demonstrated (Cushman et al J. Med. Chem.1995, 38, 2041-2049) that other electron-rich groups at the 2-positionof estradiol (propyne, propene, ethoxy) have good anti-proliferativeactivity in vitro. It is disclosed that modifications at C-2 ofestradiol such as formyl, acetyl, methanol, 1-ethanol, 2-ethanol, amino,alkylamino, dialkyl amino, methyleneamine, methylene alkyl amine andmethylene dialkylamine, and alkyl amide are be anti-proliferative andanti-angiogenic agents have reduced or removed uterotropic activity.Alkyl is defined as any carbon chain up to 6 carbons in length that isbranched or straight. Listed below in Table 1 are data of 2-modifiedestradiol derivatives in HUVEC, MDA-MB-231 and MCF7 proliferation data.The synthetic paths for preparation of these analogs can be found inPert et al Aust. J. Chem. 1989, 42, 405-419. Lovely et al TetrahedronLett. 1994, 35, 8735-8738. Gonzalez et al Steroids 1982, 40, 171-187.Nambara et al Chem. Pharm. Bull. 1970, 18, 474-480. Cushman et al J.Med. Chem. 1995, 38, 2041-2049 and methods developed in-house and arediscussed below. TABLE 1 HUVEC MDA-MB-231 MCF7 Proliferation Compound(IC₅₀ μM) (IC₅₀ μM) Index E2 NA NA 13.1 2ME2 0.5 0.9 4.4 2-methyl 10 >257.4 hydroxy-E2 2-formyl-E2 8 >25 5.4 2-acetyl-E2 18 9 4.4

All of the 2-modified analogs presented in Table 1 have significantlyless estrogenic activity (compared to estradiol) as represented by theirproliferation index in estrogen dependant MCF-7 cells. All of theseanalogs have the capacity to from a hydrogen bond with the hydroxy groupat position 3 and this may be the reason for their relatively lowestrogenic character compared to estradiol. Both the 2-methylhydroxy and2-formyl derivatives had good antiproliferative activity (IC50<10microM) in HUVEC cells, whereas the 2-acetyl had poor activity in thesame assay. In contrast, 2-methylhydroxy and 2-formyl were inactive inbreast tumor MDA-MB-231 cells while 2-acetyl E2 had good activity inthis cell line.

Although not wishing to be bound by theory, molecular modeling suggeststhat there may be a hydrogen bond that forms between the 3-hydroxy groupand the methoxy group of 2-methoxyestradiol. This interaction may beimportant for both 2-methoxyestradiol's anti-proliferative andanti-angiogenic action as well as its non-estrogenic activity. It isclaimed that any group that can be placed at position 2 of estradiol andhas the potential to form a hydrogen bond with the 3-hydroxy group is ananti-proliferative and anti-angiogenic agent that lacks estrogenicactivity.

It should be understood that in addition to the ingredients,particularly mentioned above, the formulations of this invention mayinclude other agents convention in the art having regard to the type offormulation in question, for example, those suitable for oraladministration may include flavoring agents.

Experimental Data

The following Examples refer to the compound of the general formula:

wherein:

a) R_(b) and R_(o) are independently —H, —Cl, —Br, —I, —F, —CN, loweralkyl, —OH, —CH₂—OH, —NH₂; or N(R₆)(R₇), wherein R₆ and R₇ areindependently hydrogen or an alkyl or branched alkyl with up to 6carbons;

b) R_(a) is —N₃, —C≡N, —C≡C—R, —C═CH—R, —R—C═CH₂, —C≡CH, —O—R, —R—R₁, or—O—R—R₁ where R is a straight or branched alkyl with up to 10 carbons oraralkyl, and R₁ is —OH, —NH₂, —Cl, —Br, —I, —F or CF₃;

c) Z′ is >CH, >COH, or >C—R₂—OH, where R₂ is an alkyl or branched alkylwith up to 10 carbons or aralkyl;

d) >C—R_(g) is >CH₂, >C(H)—OH, >C═O, >C═N—OH, >C(R₃)OH, >C═N—OR₃,>C(H)—NH₂, >C(H)—NHR₃, >C(H)—NR₃R₄, or >C(H)—C(O)—R₃, where each R₃ andR₄ is independently an alkyl or branched alkyl with up to 10 carbons oraralkyl;

e) R_(h1) and R_(h2) are independently H, or a straight or branchedchain alkyl, alkenyl or alkynyl with up to 6 carbons that isunsubstituted, or substituted with one or more groups selected from ahetero functionality (O—Y, N—Y or S—Y) where Y is H, Me or an alkylchain up to 6 carbons; a halo functionality (F, Cl, Br or I); anaromatic group optionally substituted with hetero, halo or alkyl; orR_(h1) and R_(h2) are independently an aromatic group optionallysubstituted with hetero, halo or alkyl, provided that both R_(h1) andR_(h2) are not H;

f) Z″ is >CH₂, >C═O, >C(H)—OH, >C═N—OH, >C═N—OR₅, >C(H)—C═N, or>C(H)—NR₅R₅, wherein each R₅ is independently hydrogen, an alkyl orbranched alkyl with up to 10 carbons or aralkyl;

and wherein all monosubstituted substituents have either an α or βconfiguration.

Lower alkyl is defined as a small carbon chain having 1-8 carbon atoms.The chain may be branched or unbranched.

EXAMPLE 1

Synthesis of 2-ME Derivatives and Modifications at the 16 Position

Synthesis of the 2-ME derivatives described herein is within thecapability of one ordinarily skilled in the art. A specific descriptionof the synthesis of the 2-ME derivatives having modifications at the 2and 6 positions and analogs discussed herein can be found in M. Cushman,H-M. He, J. A. Katzenellenbogen, C. M. Lin and E. Hamel, Synthesis,antitubulin and antimitotic activity, and cytotoxicity of2-methoxyestradiol, and endogenous mammalian metabolite of estradiolthat inhibits tubulin polymerization by binding to the colchicinebinding site, J. Med. Chem., 38(12): 2042 (1995); and M. Cushman, H-M.He, J. Katzenellenbogen, R. Varma, E. Hamel, C. Lin, S. Ram and Y. P.Sachdeva, Synthesis of analogs of 2-methoxyestradiol with enhancedinhibitory effects on tubulin polymerization and cancer cell growth, J.Med. Chem. 40(15): 2323 (1997).

The synthetic pathways used to prepare the derivatives of the estradiolderivatives modified at the 16 position of the present invention arebased on modified published literature procedures for estradiolderivatives cited earlier. Examples of the modifications are provided inExamples 2 through 23 below.

EXAMPLE 2

Preparation of 3-Benzyl-2-methoxyestradiol

2-Methoxyestradiol (10.09 g, 33.4 nmmol) and potassium carbonate (22 g,278 mmol) were suspended in anhydrous ethanol and cooled to 0° C. Benzylbromide (11.4 mL, 95.8 mmol) was added dropwise, and following theaddition, the mixture was brought to reflux for 8 h. The solution wascooled to room temperature (rt), and the solvent was removed viarotoevap. The resulting residue was diluted with approximately 200 mlwater, and washed with ethyl acetate (3×200 mL). The combined organicswere washed with water (200 mL), sodium bicarbonate (saturated (satd),200mL) and brine (200 mL). Dry with sodium sulfate, filter androto-evaporation (rotoevap). Product was dried under vacuo withoccasional gentle heating using a heat gun to give a yellowish glass(13.54 g, quanitative yield) and used without further purification.

Selected spectral data: ¹H-NMR (300 MHz, CDCl₃) δ 7.29-7.53 (m, 5H),6.88 (s, 1H), 6.65 (s, 3H), 5.11 (s, 2H), 3.87 (s, 3H), 3.7 (t, J=8 Hz,1H), 0.80 (s, 3H). FT-IR (neat) 3341, 2920, 2864, 1605, 1513, 1453,1254, 1211, 1117, 1022 cm⁻¹.

EXAMPLE 3

Preparation of 3-Benzyl-2-methoxyestrone

Oxalyl chloride (38 mmol, 19 mL, 2M, methylene chloride) was added toanhydrous methylene chloride (25 mL) and cooled to −46° C. Methylsulfoxide (5.40 mL, 76 mmol) was added dropwise, and the mixture wasstirred for 2 minutes. 3-Benzyl-2-methoxyestradiol in methylenechloride/methyl sulfoxide (10 mL/15 mL) and added within 5 minutes andthe resulting mixture was stirred for 1 h. Triethyl amine (170 mmol,23.5 mL) was added drop-wise, stirred 5 minutes and warmed to rt. Water(˜200 mL) was added and the mixture was washed with methylene chloride(3×200 mL). The combined organics were washed with water (200 mL),dilute HCl (1% aq., 200 mL), sodium carbonate (satd, 200 mL) and brine(200 mL). The organics were dried with magnesium sulfate, filtered androtoevaped to give a white solid. The solid was crystallized with hotethanol to give white crystals (9.94g, 25.5 mmol, 76% overall yield from2-methoxyestradiol).

Selected spectral data: ¹H-NMR (300 MHz, CDCl₃) δ 7.28-7.48 (m, 5H),6.86 (s, 1H), 6.66 (s, 1H), 3.88 (s, 3H), 0.94 (s, 3H). IR (neat) 2920,1731, 1519, 1202, 1012 cm⁻¹.

EXAMPLE 4

Representative Preparation of 16α-alkyl-3-benzyl-2-methoxyestrone

Lithium diisopropyl amide (2M, Aldrich, heptane/THF/ethylbenzene) wasdissolved in THF and cooled to −78° C., and 3-benzyl-2-methoxyestrone inTHF (10 mL) was added dropwise. Following addition, the mixture waswarmed to 0° C. and stirred 1 hour (h). The mixture was then cooled to−78° C. and DMPU (1 mL) followed by crotyl bromide (205 μL, 2.0 mmol)were added dropwise. The mixture was warmed to rt over 4 h. The reactionwas quenched by carefully adding water (100 mL) and washing with ethylacetate (2×100 mL). The combined organics were washed with water (100mL) and brine (100 mL). The solution was dried with magnesium sulfate,filtered and rotoevaped. The crude product was purified usinghexane/ethyl acetate (9:1) SiO₂ Biotage FLASH apparatus. 680 mg (1.53mmol) of product was obtained and approximately 121 mg (0.31 mmol) ofstarting material was recovered (90% yield based on recovered startingmaterial). Diastereomeric ratio of 16 α/β is approximately 2:1 (s H18signals at 0.88, 0.79 ppm).

Selected spectral data: ¹H-NMR (300 MHz, CDCl₃) δ 7.28-7.48 (m, 5H),6.86 (s, 1H), 6.66 (s, 1H), 5.34-5.59 (m, 2H), 5.13 (s, 2H), 3.88 (s,3H), 0.87 & 0.97 (s, total 3H, ratio 1:2).

EXAMPLE 5

Representative Preparation of 16β-alkyl-3-benzyl-2-methoxyestrone

3-Benzyl-2-methoxyestrone (1.175 g, 3.0 mmol) was dissolved in anhydrousTHF (15 mL), cooled to −78° C. and lithium diisopropyl amide (2MAldrich, heptane/THF/ethylbenzene) was added dropwise and stirred 1 h.DMPU (1 mL) followed by crotyl bromide (302 μL) were added and themixture warmed to rt over 24 h. Workup as above and purify usinghexane:ethyl acetate (4:1) SiO₂ flash column gave 492 mg purifiedproduct (1.1 mol, 37% yield).

Selected spectral data: ¹H-NMR (300 MHz, CDCl₃) δ 7.28-7.48 (m, 5H),6.86 (s, 1H), 6.66 (s, 1H), 5.62-5.34 (m, 2H), 5.13 (s, 2H), 3.89 (s,3H), 0.98 and 0.87 (s, 3H total, ratio 2:1). IR (neat) 2928, 2854, 1732,1606, 1508, 1452, 1215, 1016 cm⁻¹.

EXAMPLE 6

Representative Preparation of 16β-alkyl-3-benzyl-2-methoxyestrone

3-benzyl-16-carbomethoxy-2-methoxyestrone (0.840 g, 1.87 mmol),potassium hydride (1.5 g, 10.9 mmol, 30% mineral oil dispursion, washedin hexanes) and 18-crown-6 (120 mg, 0.4 mmol) was mixed in THF (40 mL)and refluxed for 1 h. The mixture was cooled to rt, and allyl bromide(537 μL, 6.2 mmol) was added and the mixture was refluxed for 18 h.After cooling to rt, the reaction was quenched by carefully addingapproximately 2 ml of water with stirring, then adding an additional 100mL water. This mixture was washed with ethyl acetate (2×100 mL) and thecombined organics were washed with brine (100 mL). The organics weredried with magnesium sulfate, filtered and rotoevaped. Purificationusing 85:5 hexanes:ethyl acetate SiO₂ Biotage FLASH apparatus yielded697 mg of product (1.42 mol, 76% yield).

Selected spectral data: ¹H-NMR (300 MHz, CDCl₃) δ 7.28-7.48 (m, 5H),6.85 (s, 1H), 6.66 (s, 1H), 566-5.79 (m, 1H), 5.15-5.20 (m, 2H), 5.13(s, 2H), 3.88 (s, 3H), 3.75 (s, 3H), 0.99 (s, 3H).

EXAMPLE 7

Representative decarboxylation of16-alkyl-16-carbomethoxy-3-benzyl-2-methoxyestrone

16-allyl-16-carbomethoxy-3-benzyl-2-methoxyestrone (697 mg, 1.42 mmol),lithium chloride (1.15 g, 27 mmol), water (485 μL, 27 mmol) weredissolved in DMF (63 mL) and refluxed for 20 h. Cool to rt, add 1N HCl(100 mL) and wash with ether (2×100 mL) the combined organics werewashed with water (100 mL), and brine 100 mL), dry with magnesiumsulfate, filter and rotoevap. Purification by 85:15 hexanes:ethylacetate SiO₂ Biotage Flash apparatus gave 271 mg product and 189 mgrecovered starting material. Starting material was resubjected to thereaction (308 mg LiCl, 132 μL, water, 17 mL DMF) for 28 h and worked upas above to give 130 mg product. Overall yield for reaction was 66% (401mg, 0.93 mmol).

Selected spectral data: ¹H-NMR (300 MHz, CDCl₃) δ 7.28-7.48 (m, 5H),6.85 (s, 1H), 6.65 (s, 1H), 5.69-5.88 (m, 1H), 5.13 (s, 2H), 5.00-5.08(m, 2H), 5.88 (s, 3H), 0.98 nd 0.88 (s, total 3H, ratio 1:1.4). FT-IR(neat), 2925, 2855, 1726, 1514, 1214, 1103 cm⁻¹.

EXAMPLE 8

Preparation of 16-methane-dimethylenamine-3-benzyl-2-methoxyestrone

3-benzyl-2-methoxyestrone (1.51 g, 3.87 mmol) was suspended intert-butoxy bis(dimethylamino)methane (1.64 mL, 8.13 mmol) and heated inan oil bath (155° C.) for 1.5 h, during which time the steroiddissolved. The reaction mixture was cooled to rt, and poured into icewater (100 mL) and washed with methylene chloride (2×100 mL). Theorganics were washed with brine (100 mL) dried with magnesium sulfate,filtered and rotoevaped to give product which was used without furtherpurification (1.82 g, quanitative yield).

Selected spectral data: ¹H-NMR (300 MHz, CDCl₃) δ 7.23-7.47 (m, 5H),6.87 (s, 1H), 6.64 (s, 1H), 5.12 (s, 2H), 3.88 (s, 3H), 3.07 (s, 6H),0.91 (s, 3H).

EXAMPLE 9

Preparation of 16-carbomethoxy-3-benzyl-2-methoxy estrone

3-Benzyl-2-methoxyestrone (1.6113 g, 2.978 mmol) was dissolved in THF(15 mL), cooled to −78° C. and lithium diisopropyl amide (2M, Aldrich,Heptane/THF/ethylbenzene) was added dropwise and stirred for 1 h. Methylcyanoforrnate (237 μL, 3 mmol) in DMPU (1 mL) was added and the mixturewarmed to rt over 18 h. Water (100 ml) was carefully added, and themixture was washed with ethyl acetate (3×100 mL) and the combinedorganics were washed with brine (100 mL), dried with sodium sulfate,filtered and rotoevaped. Final purification of product usinghexane:ethyl acetate (85:15) then switching to hexane:ethyl acetate(75:25) SiO₂ flash column yielded 806 mg product (1.8 mmol, 60%).

Selected spectral data: ¹H-NMR (300 MHz, CDCl₃) δ 7.28-7.48 (m, 5H),6.85 (s, 1H), 6.66 (s, 1H), 5.13 (s, 2H), 3.88 (s, 3H), 3.78 (s, 3H),3.23 (dd, J =9, 10 Hz, 1H), 1.0 (s, 3H). FT-IR (neat) 2929, 2860, 1750,1723, 1604, 1508, 1211, 1014 cm¹.

EXAMPLE 10

Representative Procedure for Preparation of16-alkyl-3-benzyl-2-methoxyestra-17β-diol

16α-crotyl-3-benzyl-2methoxyesttone (680 mg, 1.53 mmol) was dissolved inanhydrous THF (10 mL), and cooled to −78° C. Lithium aluminum hydride(3.06 mmol, 116 mg) was added and the solution was stirred for 2 h. Thereaction was quenched by carefully adding water (2 mL) and warming tort, then adding additional 50 mL portion of water. The mixture waswashed with ethyl acetate (2×50 mL) and the combined organics werewashed with water (50 mL), brine (50 mL), dried with magnesium sulfate,filtered and rotoevaped. The mixture was purified with 3:1 hexane:ethylacetate SiO₂ Biotage FLASH apparatus to give 500 mg purified product(1.12 mmol, 73% yield).

Selected spectral data: ¹H-NMR (300 MHz, CDCl₃) δ 7.28-7.48 (m, 5H),6.87 (s, 1H), 6.64 (s, 1H), 5.47-5.56 (m, 2H), 5.12 (s, 2H), 3.88 (s,3H), 3.8 (d, J=9 Hz) and 3.33 (d, J=8Hz) total 1H, ratio 1:1.7, 0.84 and0.81 (s, 3H total).

EXAMPLE 11

Preparation of 16-methanol-3-benzyl-2-methoxyestradiol

Reaction procedure and work up as above, (used 806 mg, 1.8 mmol16-carbomethoxy-3-benzyl-2-methoxyestrone), except warm to rt for 2 hbefore quenching. Purify final product with 3:2 hexane:ethyl acetateSiO₂ flash column. Obtain 304 mg β isomer, 51 mg α isomer which wereseparated by chromatography. Selected spectral data: ¹H-NMR (300 MHz,CDCl₃) δ Major isomer 7.28-7.48 (m, 5H), 6.87 (s, 1H), 6.64 (s, 1H),5.12 (s, 2H), 3.97 (d, J=10 Hz), 3.88 (s and obscured d, 4H), 3.67 (dd,J=4, 7Hz, 1H), 0.87 (s, 3H). Minor isomer 7.28-7.47 (m, 5H), 6.86 (s,1H), 6.64 (s, 1H), 3.88 (s, 3H), 3.83 (d, J=14Hz, 1H), 3.69 (t, J=9Hz,1H), 3.54 (d, J=7Hz, 1H), 0.87 (s, 3H).

EXAMPLE 12

Representative Debenzylation of 16-alkyl-3-benzyl-2-methoxyestradiol

16α-crotyl-3-benzyl-2-methoxyestradiol (500 mg, 1.12 mmol) was dissolvedin ethyl acetate (25 mL) in Parr reaction bottle. The bottle was flushedwith argon, and Pd/C (10%, 2.5 g) was added. The bottle was fitted to aParr hydrogenator, filled and purged with hydrogen five times,pressurized to 50 psi, and agitated for 24 h. The mixture was filteredthrough a celite pad, rotoevaped and purified with a 3:1 hexane ethylacetate SiO₂ flash column. Obtain 358 mg product (1.0 mmol, 89%).

Selected spectral data: ¹H-NMR (300 MHz, CDCl₃) δ 6.81 (s, 1H), 6.66 (s,1H), 3.87 (s, 3H), 3.76 (d, J=10 Hz) and 3.29 (d, J=8Hz) (total 1H,ratio 1:2), 0.82 and 0.79 (s, 3H). FT-IR (neat) 3245, 2914, 1606, 1523,1414, 1258, 1028 cm⁻¹. Analysis calculated (Anal. Calcd) for C₂₀H₃₄O₃:C, 77.44; H, 9.56. Found: C, 76.64; H, 9.51.

EXAMPLE 13

16β-methyl-2methoxyestradiol

Selected spectral data: ¹H-NMR (300 MHz, CDCl₃) δ 6.81 (s, 1H), 6.66 (s,1H), 3.87 (s, 3H), 3.73 (d, J=10 Hz) and 3.23 (d, J=8 Hz) (total 1H,2:1), 0.83 and 0.81(s, 3 H total). Anal. Calcd for C₂₀H₂₈O₃, 1/4 H₂O: C,74.85; H, 8.95. Found: C, 74.93; H, 8.94.

EXAMPLE 14

16α-methyl-2methoxyestradiol

Selected spectral data: ¹H-NMR (300 MHz, CDCl₃) δ 6.81 (s, 1H), 6.66 (s,1H), 3.87 (s, 3H), 3.23 (d, J=7 Hz) (s, 1H), 0.81 (s, 3 H). Anal. Calcdfor C₂₀H₂₈O₃, 1/4 H₂O: C, 74.85; H, 8.95. Found: C, 74.98; H, 8.65.

EXAMPLE 15

Racemic 16-ethyl-2-methoxyestradiol

Selected spectral data: ¹H-NMR (300 MHz, CDCl₃) δ 6.82 (s, 1H), 6.66 (s,1H), 3.88 (s, 3H), 3.76 (d, J=9 Hz) and 3.30 (d, J=10 Hz), (1H total,ratio 1:1), 0.83 and 0.79 (s, 3H total). FT-IR (neat) 3214, 2918, 1605,1522, 1229, 1201, 1024 cm⁻¹. Anal. Calcd for C₂₁H₃₀O₃: C, 76.33; H,9.15. Found: C, 76.18; H, 9.16.

EXAMPLE 16

16α-n-propyl-2-methoxyestradiol

Selected spectral data: ¹H-NMR (300 MHz, CDCl₃) δ 6.81 (s, 1H), 6.66 (s,1H), 5.43 (s, 1H), 3.87 (s, 3H), 3.29 (t, J=7 Hz, 1H), 0.95 (t, J=7 Hz,3H), 0.83 and 0.80 (s, total 3H, ratio 7.3:1). Anal. Calcd for C₂₂H₃₂O₃:C, 76.69; H, 9.37. Found: C, 76.55; H, 9.44.

EXAMPLE 17

16β-n-propyl-2-methoxyestradiol

Selected spectral data: ¹H-NMR (300 MHz, CDCl₃) δ 6.81 (s, 1H), 6.66 (s,1H), 3.87 (s, 3H), 3.76 (d, J=10 Hz) and 3.29 (t, J=7 Hz) (total 1H,ratio 2:1), 0.95 (t, J=7 Hz, 3H), 0.83 and 0.80 (s, total 3H). FT-IR(neat) 3411, 2923, 1504, 1446, 1267, 1202, 1118, 1024 cm⁻¹. Anal. Calcdfor C₂₂H₃₂O₃, 1/4 H₂O: C, 75.71; H, 9.39. Found: C, 75.61; H, 9.33.

EXAMPLE 18

16β-n-butyl-2-methoxyestradiol

Selected spectral data: ₁ H-NMR (300 MHz, CDCl₃) δ 6.81 (s, 1H), 6.66(s, 1H), 5.43 (s, 1H), 3.88 (s, 3H), 3.76 (d, J=10 Hz) 3.29 (d, J=8 Hz)(total 1H, ratio 2.6:1), 0.83 and 0.80 (s, total 3H). FT-IR (neat) 3221,2921, 1594, 1504, 1416, 1265, 1200, 1021 cm⁻¹. Anal. Calcd for C₂₃H₃₄O₃:C, 77.04; H, 9.56. Found: C, 77.06; H, 9.65.

EXAMPLE 19

16β-isobutyl-2-methoxyestradiol

Selected spectral data: ¹H-NMR (300 MHz, CDCl₃) δ 6.81 (s, 1H), 6.66 (s,1H), 5.43 (s, 1H), 3.88 (s, 3H), 3.77 (dd, J=9, 10 Hz) and 3.26 (t, J=7Hz) (total 1 H, ratio 2:1), 0.84 and 0.80 (s, total 3H). IR (neat) 3525,2913, 1506, 1258, 1202, 1026 cm⁻¹. Anal. Calcd for C₂₂H₃₀O₃: C, 76.69;H, 9.37. Found: C, 76.82; H, 9.47.

EXAMPLE 20

16β-methyl(dimethyl amine)-2-methoxyestradiol

Selected spectral data: ¹H-NMR (300 MHz, CDCl₃) δ 6.81 (s, 1H), 6.65 (s,1H), 3.88 (s) and 3.85 (obscured d) (total 4H), 2.28 (s, 6H), 0.87 (s,3H). Anal. Calcd for C₂₂H₃₃O₃N, 1/4 H₂O: C, 72.59; H, 9.28; N, 3.85.Found: C, 72.80; H, 9.17; N, 3.66.

EXAMPLE 21

16,β-methanol-2-methoxyestradiol

Selected spectral data: ¹H-NMR (300 MHz, CDCl₃) δ 6.78 (s, 1H), 6.61 (s,1H), 3.92 (d, J=11Hz, 1H), 3.84 (s, 3H), 3.80 (d, J=10 Hz, 1H), 3.63 (d,J=8, 11Hz, 1H), 0.83 (s, 3H). FT-IR (neat) 3283, 3091, 2919, 1602, 1513,1445, 1204, 1119, 1013 cm⁻¹. Anal. Calcd for C₂₀H₂₈O₄: C, 72.25; H,8.49. Found: C, 72.24; H, 8.48.

EXAMPLE 22

16β-methanol-2-methoxyestradiol

Selected spectral data: ¹H-NMR (300 MHz, CDCl₃) δ 6.77 (s, 1H), 6.61 (s,1H), 3.84 (s, 3H), 3.84 (dd, J=7, 8 Hz, 1H), 3.61 (dd, J=9, 11 Hz, 1H),3.45 (d, J=8 Hz, 1H), 0.83 (s, 3H).

EXAMPLE 23

MDA-MB-231 In Vitro Cellular Proliferation Inhibition

MDA-MB-231 Cells and Culture Conditions

FIG. 1 illustrates the antiproliferative activity in cells and tumor by2-methoxyestradiol compounds of the present invention which are modifiedat the 16 position.

MDA-MB-231 human breast carcinoma cells were grown in DMEM containing10% FCS (Hyclone Laboratories, Logan Utah) and supplemented with 2 mML-Glutamine, 100 units/ml penicillin, 100 μg/ml streptomycin (IrvineScientific, Santa Anna, Calif.).

Proliferation Assays

MDA-MB-231 cells were plated at 5000 cells/ml in 96 well plates. Afterallowing the cells to attach overnight, the appropriate fresh media wereapplied containing differing concentrations of 2-ME2 or derivativesthereof, as described below. Drug was dissolved in DMSO (Sigma, St.Louis, Mo.) and added to the wells in a volume of 200 μl. Cells wereincubated for two days at 37° C.; at 32 h BrdU was added. BrdU cellproliferation assay (a nucleotide analogue with a fluorescein tag thatis incorporated into DNA) was performed as described by the manufacturer(Roche). Each condition was prepared in triplicate and the experimentswere carried out a minimum of two times. Results are presented and means±SE.

EXAMPLE 24

HUVAC In Vitro Cellular Proliferation Inhibition

HUVAC Cells and Culture Conditions

HUVAC Cells were Grown in EGM (Clonetics)

Proliferation Assays

HUVEC cells were plated at 5000 cells/ml in 96-well plates. Afterallowing the cells to attach overnight, the cells were washed with PBSand incubated in the absence of growth factor for 24 h (EBM, 2% FCS,Clonetics). Cells were treated with increasing concentrations of drug inEBM containing 2% FCS and 10 ng.ml bFGF for 48 h at 37° C. Drugpreparation, volumes added and BrdU proliferation assay were performedas indicated above.

Results

The breast cancer cell line activities and the cell panels mostsensitive to selected analogs are shown in Table 2. TABLE 2 α/β ratio atHUVEC MDA-MB-231 R position 16 IC₅₀ (μM) IC₅₀ (μM) H N/A 0.5 0.9 methyl(—CH₃) All alpha <0.5 <0.5 methyl 1:2 1.3 5 ethyl (—CH₂CH₃₎ 1:1 2 3n-propyl 7.3:1 6 >50 (—CH₂CH₂CH₃) n-propyl 1:2 9 36

1:2 7.5 40 n-butyl 2:1 25 82 (—CH₂CH₂CH₂CH₃) n-butyl 1:2.6 9 39 methanolAll alpha 15 22 (—CH₂OH) methanol All beta 5 50

All beta 9 22

2-Methoxyestradiol is a potent anti-angiogenic and anti-tumor agent. Inorder to assess the biological activity of modifications at position 16,the anti-proliferative activity of these analogs was evaluated on humanumbilical vein endothelial cells (HUVEC) and breast carcinoma cell line,MDA-MB-231 as models for the anti-angiogenic and anti-tumor activity,respectively. It was found that a moderate decrease (approximately 18fold) in antiproliferative activity occurs as steric bulk increased(note trend from R=Et to R=Bu). The most active compound in this seriesis 16α-methyl, which has greater activity than 2-methoxyestradiol.

The MDA-MB-231 tumor cell line, has a much greater sensitivity tosubstitutions at position 16 compared to HUVEC cells. Any group atposition 16 larger than ethyl has a significant decrease inantiproliferative activity (IC₅₀>22 μM). Of the active compounds,16α-methyl has better activity than 2-methoxyestradiol, whereas16β-methyl (which is a 1:2 mixture of α:β, so the presence of the αisomer may account for this activity) has about 5-fold less activitythan 2-methoxyestradiol, and racemnic 16-ethyl has about a 3-fold dropin activity compared to 2-methoxyestradiol.

These data suggest that it is possible to design compounds that areselective anti-angiogenic agents. For example, 16α-propyl is greaterthan ten-fold less active in inhibiting tumor growth while it has goodactivity inhibiting endothelial cell proliferation. Other examplesinclude: 16β-propyl (4-fold difference), 16β-i-butyl (5-folddifference), 16β-n-butyl (4-fold difference) and 16β-methanol (10-folddifference). Additionally, a small alkyl group at position 16 can beadded without significantly impacting the anti-proliferative activity ofthe molecule.

All of the publications mentioned herein are hereby incorporated byreference in their entireties. The above examples are merelydemonstrative of the present invention, and are not intended to limitthe scope of the appended claims.

1-2. (canceled)
 3. The method of claim 10, wherein: R_(h1) and R_(h2)are independently H and Et.
 4. The method of claim 10, wherein: R_(h1)and R_(h2) are independently H and n-Pr.
 5. The method of claim 10,wherein: R_(h1) and R_(h2) are independently H and i-Bu.
 6. The methodof claim 10, wherein: R_(h1) and R_(h2) are independently H and CH₂OH.7. The compound method of claim 10, wherein: R_(h1) and R_(h2) areindependently H and n-Bu.
 8. The method of claim 10, wherein: R_(h1) andR_(h2) are independently H and Me.
 9. The method of claim 10, wherein:R_(h1) and R_(h2) are independently H and (CH₂)_(n)—C(Me)₂.
 10. A methodof inhibiting angiogenesis in a human or an animal comprisingadministering to the human or animal an angiogenesis inhibiting amountof a compound of the formula:

wherein: a) R_(a) is —N₃, —C≡N, —C≡C—R, —C═CH—R, —R—C═CH₂, —C≡CH, —O—R,—R—R₁, or —O—R—R₁ where R is a straight or branched alkyl with up to 10carbons or aralkyl, and R₁ is —OH, —NH₂, —Cl, —Br, —I, —F or CF₃; b) Z′is >CH or >C—R₂—OH, where R₂ is an alkyl or branched alkyl with up to 10carbons or aralkyl; c) >C—R_(g) is >CH₂, >C(H)—OH, >C═O, >C═N—OH,>C(R₃)OH, >C═N—OR₃, >C(H)—NH₂, >C(H)—NHR₃, >C(H)—NR₃R₄, or>C(H)—C(O)—R₃, where each R₃ and R₄ is independently an alkyl orbranched alkyl with up to 10 carbons or aralkyl; d) R_(h1) and R_(h2)are independently H, or a straight or branched chain alkyl, alkenyl oralkynyl with up to 6 carbons that is unsubstituted, or substituted withone or more groups selected from a hetero functionality (O—Y, N—Y orS—Y) where Y is H, Me or an alkyl chain up to 6 carbons; a halofunctionality (F, Cl, Br or I); an aromatic group optionally substitutedwith hetero, halo or alkyl; or R_(h1) and R_(h2) are independently anaromatic group optionally substituted with hetero, halo or alkyl,provided that both R_(h1) and R_(h2) are not H; and wherein allmonosubstituted substituents have either an α or β configuration.